EP0718408A2 - Méthode sensible pour détecter des acides nucléiques - Google Patents
Méthode sensible pour détecter des acides nucléiques Download PDFInfo
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- EP0718408A2 EP0718408A2 EP95118269A EP95118269A EP0718408A2 EP 0718408 A2 EP0718408 A2 EP 0718408A2 EP 95118269 A EP95118269 A EP 95118269A EP 95118269 A EP95118269 A EP 95118269A EP 0718408 A2 EP0718408 A2 EP 0718408A2
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- nucleic acid
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/682—Signal amplification
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6823—Release of bound markers
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Definitions
- the invention relates to a method for the particularly sensitive detection of nucleic acids by hybridization of a probe nucleic acid with a target nucleic acid, degradation of the hybridized part of the probe nucleic acid and detection of the cleavage product and a set of reagents suitable for this.
- nucleic acids After it was found that the use of the specific information of nucleic acids could have great advantages in the detection of infection parameters and genetic conditions, attempts were made to make nucleic acids the subject of test procedures. In many cases, however, the amplification of the nucleic acid sequences is necessary for detection in order to be able to generate a sufficient signal in the detection method used. For this purpose, methods for increasing a target sequence as well as methods for increasing a nucleic acid sequence independent of the target sequence have been proposed. An example of an increase in the target sequence is the polymerase chain reaction (PCR) described in EP-A-0 201 184, in which the two strands of the target nucleic acid are amplified in an in vitro replication reaction.
- PCR polymerase chain reaction
- the temperature of the reaction is changed cyclically in order to denature a double-stranded target nucleic acid, to enable hybridization with initiator molecules (primers) and the subsequent DNA synthesis (extension of the primers) and to repeat these steps. If all steps of the PCR run optimally, there is an exponential amplification of nucleic acids. In practice, however, the amplification factor is often greatly reduced.
- the target-sequence-specific signal amplification is not used to increase the nucleic acid to be detected, but is only aimed at the specific identification of the target sequence.
- a signal is amplified depending on the presence of the target sequence.
- Such a method is e.g. B. in WO 89/09284 and WO 89/10415.
- CPR cycling probe reaction
- a labeled, chimeric DNA-RNA-DNA probe molecule is used, which hybridizes with a target DNA molecule. The hybridization creates an RNA-DNA hybrid, which is a substrate for RNAse H.
- the probe molecule Since this enzyme specifically degrades the RNA portion of the hybrid, the probe molecule is cleaved and the resulting fragments diffuse from the target sequence due to the lower melting temperature. The target molecule can then hybridize with another probe molecule and the cycle is repeated. The fragments of the probe molecule are detected via the label on them.
- the aim of the present invention was to increase the sensitivity of methods in which a target nucleic acid is hybridized with a probe molecule and the degradation of the probe molecule is used to determine the target nucleic acid.
- the invention also relates to a method for amplifying short nucleic acid sequences with the aid of longer nucleic acid sequences which contain the shorter nucleic acid sequences, a method for the sensitive detection of a target nucleic acid A and kits for carrying out these methods.
- Target nucleic acids are understood to mean nucleic acids that are to be detected either directly or indirectly.
- the target nucleic acid is identified with the letter A.
- Methods for the direct detection of target nucleic acids are understood to mean those in which the nucleic acids are already available for the method steps according to the invention.
- Indirect methods are understood to mean those in which the target nucleic acids are subjected to a pretreatment step or are provided by them.
- Such pretreatment steps can, for example, isolate the nucleic acids, treat with reagents, e.g. B. restriction enzymes, or pre-amplification. Reverse transcription of RNA can also be viewed as pretreatment.
- the target nucleic acid can therefore be of any, for example viral, bacterial or cellular origin. It can be present in solution, suspension, but also fixed to solids or in cell-containing media, cell swabs, fixed cells, tissue sections or fixed organisms.
- the nucleic acid is preferably in solution.
- nucleic acids are understood to mean nucleic acids that can hybridize with the corresponding nucleic acid or hybridize with a nucleic acid that is complementary to the corresponding nucleic acid.
- the detection methods for nucleic acids usually begin with a step for making the target nucleic acid available with appropriate reagents. Changes in pH (alkaline), heat, repetition of extreme temperature changes (freezing / thawing), changes in physiological growth conditions (osmotic pressure), exposure to detergents, chaotropic salts or enzymes (e.g. proteases, lipases) alone or in Combination contribute to the release of the nucleic acids. Since the method according to the invention is very sensitive and selective, even small amounts of nucleic acids can be detected in the presence of other substances, for example proteins, cells, cell fragments, but also non-detectable nucleic acids.
- other substances for example proteins, cells, cell fragments, but also non-detectable nucleic acids.
- Suitable target nucleic acids are, for example, ribonucleic acids and deoxyribonucleic acids.
- the nucleic acids can also be modified, for example by the processing steps previously carried out.
- Deoxyribonucleic acid (DNA) is particularly preferred as the target nucleic acid.
- the method according to the invention is a special embodiment of a test based on a hybridization event, in particular the target sequence-specific signal amplification.
- the basic principles of tests based on hybridization events are known to those skilled in the field of nucleic acid diagnostics. Insofar as experimental details are not set out below, the full content of this is "Nucleic acid hybridization", publisher B.D. Hames and S.J. Higgins, IRL Press, 1986, e.g. B. in Chapters 1 (Hybridization Strategy), 3 (Quantitative Analysis of Solution Hybridization) and 4 (Quantitative Filter Hybridization), Current Protocols in Molecular Biology, Edt. F.M. Ausubel et al., J.
- nucleoside triphosphates as described in EP-A-0 324 474, the chemical synthesis of modified and unmodified oligonucleotides, the cleavage of nucleic acids by means of restriction enzymes, the selection of hybridization conditions by means of which specificity can be achieved, which depends, among other things, on the extent of the complementarity between the nucleic acids to be hybridized, their GC content and their length, and the formation of nucleic acids from nucleoside triphosphates with the aid of polymerases, optionally using so-called primers.
- a label in the sense of the present invention consists of a directly or indirectly detectable group.
- Directly detectable groups are, for example, radioactive ( 32 P), colored or fluorescent groups or metal atoms.
- Indirectly detectable groups are, for example, immunologically or enzymatically active compounds such as antibodies, antigens, haptens or enzymes or enzymatically active partial enzymes. These are detected in a subsequent reaction or reaction sequence. Haptens are particularly preferred, since nucleoside triphosphates labeled with them can generally be used particularly well as substrates for polymerases and a subsequent reaction with a labeled antibody against the hapten or the haptenized nucleoside is easily carried out can be.
- nucleoside triphosphates are, for example, bromine nucleoside triphosphates or digoxigenin, digoxin, biotin or fluorescein-coupled nucleoside triphosphates.
- the steroids mentioned in EP-A-0 324 474 and their detection have proven to be particularly suitable.
- Nucleoside triphosphates are ribo (rNTP) - or deoxyribo-nucleoside triphosphates (dNTP).
- nucleic acids with a longer sequence are generated from a plurality of nucleic acids with a short sequence, which can act as primers.
- a longer sequence is a sequence which is 1 or more, preferably 5-1000, particularly preferably 8-100, nucleobase units (eg mononucleotides) longer than the short nucleic acid.
- Two conditions are attached to the short nucleic acid. First, it must be able to form hybrids with nucleic acids through base-base interactions. Such sequences contain in particular nucleic acids which are built up from the natural nucleotide building blocks.
- sequences can also be nucleic acid analogs or modified nucleic acids, e.g. B. act nucleic acids that still have the hybridization properties of a nucleic acid, but have no phosphate sugar chain portion, or have non-natural bases. Molecules of WO 92/20702 or WO 86/05518 are particularly suitable for this.
- the second condition for the sequence is that it can be used as a primer as an extension reaction.
- extension reactions are in particular the polymerase-dependent extension of the primers by mononucleotide units using mononucleoside triphosphates.
- the 3 'end of the primer in the state of hybridization with a template nucleic acid is extended by mononucleotides, so that the extension product is essentially complementary to the corresponding position on the template nucleic acid.
- the primers or short nucleic acid can be generated in any manner according to the invention, but preference is given to cleaving a longer nucleic acid, in particular by partial enzymatic degradation of this nucleic acid.
- the primers generated in the step according to the invention preferably have a length of more than 12 bases.
- the primers preferably contain a sequence of 12 to 35 bases, which is essential for further processing of the primers (e.g. hybridization and enzymatic extension).
- the primers, particularly at their 5 'end, can contain many other nucleotides or non-nucleotide parts of the molecule if desired.
- the primers formed in the first step or parts thereof e.g. B. a partial sequence amplified.
- the amplification of the primer preferably also includes the degradation of a longer molecule.
- the amplification is particularly preferably carried out by extension, e.g. B. a longer sequence compared to the primer (short sequence), the primer on a template nucleic acid C (to a longer nucleic acid), hybridization of an amplifier probe D with the extension product of the primer and degradation (cleavage) of the amplifier probe to produce a new primer molecule. Because the extension product of this primer is again available for cleaving an amplifier probe, the number of primer molecules increases with each cleavage event. In addition, the extension of the primers formed by the cleavage again leads to the formation of extension products which are again available for the cleavage of an amplifier probe.
- the amount of primers or extension products or further intermediate products produced is a measure of the presence of target nucleic acids A in a sample.
- the method according to the invention is therefore a method in which both a once-generated extension product of a primer is used again for the cyclical generation of one or more primers, and a once-generated primer is used again for the production of an extension product.
- a longer nucleic acid is understood here to mean a nucleic acid which contains an inactive priming part and a part which, when it is broken down, leads to activation of the primer. After activation of the primer, it is available to carry out an extension reaction.
- One aspect of the invention is concerned with the amplification of short nucleic acids, in particular primers, by breaking down the longer nucleic acids to form the primers.
- the probe nucleic acids B are also such longer nucleic acids.
- Short nucleic acids in the sense of the invention are the degradation products of the longer nucleic acids mentioned above.
- the short nucleic acids are nucleic acids that can act as primers, i.e. H. can be extended by an extension area.
- Suitable probe nucleic acids B are molecules which contain two parts B1 and B2 which are connected to one another.
- Part B1 is characterized in that it can hybridize with the target nucleic acid or a part thereof. This part is sufficiently complementary to this.
- part B1 must be able to be cleaved if it is in a form hybridized with the target nucleic acid. Degradation or cleavage is to be understood here as the division of part B1 into two or more parts that are no longer bound to one another or the cleavage of part B1 from B2, the target nucleic acid preferably not being cleaved.
- Part B1 can therefore, for. B. contain ribonucleotide or abasic sequences.
- part B1 contains two, particularly preferably four or more monoribonucleotide units linked to one another in the manner customary in nucleic acids, while the part of the target nucleic acid complementary to this represents a deoxyribonucleic acid.
- the probe nucleic acid B can be cleaved in part B1 by bringing the hybrid formed into contact with RNAse H. In the presence of abasic sequences, degradation can take place using AP endonuclease.
- a cleavage of the target nucleic acid may also be accepted, ie one primer is generated for each target molecule instead of several.
- At least part of the hybridizable part B1 is split. This forms a cleavage product B ', which contains the nucleic acid-specific part B2 which is not cleavable in the hybrid of B with the target nucleic acid and preferably does not hybridize with the target nucleic acid, and possibly parts of the part B1 which was originally hybridized with the target nucleic acid.
- the conditions for the hybridization of the target nucleic acid with the probe nucleic acid are preferably chosen such that selective hybridization is also carried out the probe nucleic acid takes place via part B1 with the target nucleic acid, but an unselective hybridization of the probe nucleic acid with other, undetectable nucleic acids of the sample no longer takes place.
- the fragments resulting from the cleavage of the probe nucleic acid, including the cleavage product B ' are shorter than the original probe nucleic acid and will therefore no longer be able to form a stable hybrid with the target nucleic acid under the selected conditions. They will therefore release the target nucleic acid A under the selected conditions. This is ready for hybridization with another probe nucleic acid.
- fragments B ' can also be formed, which either only contain part B2 alone or additionally contain residues of part B1. This depends on the conditions used in each case.
- a nucleic acid-specific part of a nucleic acid is understood below to mean a sequence which can hybridize with a sequence other than the target nucleic acid, a specific hybridization taking place under the selected conditions, i. H. there is no hybridization relevant to the overall process with other nucleic acids present in the reaction mixture that are not involved in the respective reaction step.
- Typical nucleic acid-specific parts are part B2 of the probe nucleic acid, part C2 of the template nucleic acid and part D2 of the amplifier probe.
- the nucleic acid-specific part B2 of the probe nucleic acid which preferably does not hybridize with the target nucleic acid must meet the condition that it is not degraded under the conditions of the target sequence-specific degradation of part B1. In addition, it should be able to hybridize with the template nucleotide defined later.
- the sequence of part B2 can in principle be chosen freely. However, it must be taken into account here that the hybridization of the cleavage product B 'with the template oligonucleotide is made more difficult by a complementarity with the target nucleic acid, as a result of which the sensitivity to the optimal case is likely to be reduced.
- B2 can also be a ribonucleic acid, deoxyribonucleic acid or modified nucleic acid.
- part B2 preferably does not represent a ribonucleic acid which can be cleaved under these conditions, preferably it represents deoxyribonucleic acid.
- B2 can also be a modified ribonucleic acid such that it no longer from one RNAse can be cleaved.
- nucleic acid analogs which, although they still have the hybridization properties of a nucleic acid, have no phosphate-sugar chain portion or non-natural bases.
- PNA molecules from WO 92/20702 or molecules from WO 86/05518 are particularly suitable for this.
- the preferred condition that this portion should not hybridize with the target nucleic acid relates in particular to the conditions applied during the hybridization of the probe nucleic acid with the target nucleic acid.
- Part B2 is preferably selected such that it does not hybridize with non-detectable nucleic acids of the sample. However, part B2 is said to contain a sequence which can hybridize with a template nucleic acid C.
- the template nucleic acid C contains a part C2 which can hybridize with the cleavage product B 'and in particular its nucleic acid-specific part B2 or a part thereof and any residues of B1 which may still be present.
- Suitable as template nucleic acid C are all molecules which permit such hybridization, i.e. H. Nucleic acids that are made up of the natural nucleotide building blocks, but also nucleic acid analogs that are made up of non-naturally occurring building blocks or contain such building blocks and can act as a template / template.
- the template nucleic acid should be stable to degradation under the chosen conditions.
- the template nucleic acid is particularly preferably deoxyribonucleic acid.
- the template nucleic acid has a part C1 which cannot hybridize with part B1 or residues of part B1 of the probe nucleic acid which are still present. This part C1 particularly preferably does not form any hybrids which are stable under the selected conditions, even with nucleic acids not to be detected in the sample and the target nucleic acid itself.
- Part C1 is preferably in the 5 ′ direction, as seen from part C2.
- cleavage product or products B ' can hybridize with the template nucleic acid in such a way that B' also hybridizes with the template nucleic acid at the end resulting from cleavage.
- the hybrid of cleavage product B 'and template nucleic acid C is extended by a nucleic acid part B3 complementary to part C1. Therefore, B 'must be able to serve as a primer, i.e. H. It must be possible to extend the die at the 3 'end. This can be done in any way, but preferably by enzymatic extension.
- the condensation of mononucleoside triphosphates using C1 as template nucleic acid is particularly preferably understood as an enzymatic extension.
- the conditions used are known to the person skilled in the art.
- the extension can be carried out using DNA polymerase from Thermus aquaticus according to EP-A-0 258 018.
- the preferred mononucleoside triphosphates are dNTPs.
- the cleavage product B acts as a primer and short nucleic acid, which is extended.
- Another possibility of the enzymatic extension is the use of a ligase. Care should be taken to ensure that the extension product formed is stable under the conditions of probe nucleic acid cleavage. This is achieved, for example, if both the primer and the extension piece are DNA if RNAse H is used as a means for cleaving the probe nucleic acid.
- any hybrids formed from uncleaved probe nucleic acid B and template nucleic acid C are not prolonged, since e.g. B. the 3 'end of the probe nucleic acid does not hybridize with the template nucleic acid.
- the extension of uncleaved probe nucleic acids B on the target nucleic acid as a template does not influence the generation of primer B 'and can be prevented by blocking the 3' end of probe nucleic acid B, for example by using a dideoxynucleotide as the last nucleotide.
- the amplification of the primers is achieved in that, after denaturing the hybrid of template nucleic acid and the primer B 'extended by B3, the formation of a region B3 for hybridizing B3 with an amplifier probe D is used.
- An amplifier probe D is understood to mean a nucleic acid which contains a part D1 which is over its entire length or partially complementary to B3, that is to say the part of the extension product of the extension product which is formed by extension Primers (cleavage product B '). The part newly attached to the primer is referred to below as B3.
- An essential feature of the amplifier probe D is that it can in turn be split in its part D1. It is preferably designed in such a way that the cleavage conditions for the amplifier probe and the probe nucleic acid B are essentially identical with regard to the reagents used. Therefore D1 is also preferably a ribonucleic acid.
- the amplifier probe D contains a part D2 which fulfills the conditions of a nucleic acid-specific sequence.
- the special amplification effect of the method results from the fact that the degradation of the amplification probe directly or indirectly creates a cleavage product which can hybridize with a template nucleic acid and thus (as a primer) can initiate a new amplification cycle.
- amplification probes can bind one after the other under suitable conditions per extension product, so that several cleavage products are formed, since analogously to probe nucleic acid B, the cleavage products do not form a stable hybrid with the extension product.
- the amplifier probe is selected so that the further part D2 is the full length or only partially complementary to C2 and thus essentially identical to the originally formed fission products B '.
- the cleavage products D 'formed in the degradation reaction of D can be used again in the extension reaction using the template nucleic acid C as a template.
- the amplifier probe D is selected so that it contains, in addition to part D1, a nucleic acid-specific part D2, which, however, is not complementary to C2 and does not hybridize with other nucleic acids contained in the reaction mixture, with the exception of another Template nucleic acid E which has a part E2 which can be hybridized with D2 and a part E1 which acts as a template for extending D '.
- the newly formed part D3 is analogous to part B3, preferably after hybrid separation, ready to cleave a further amplifier probe F.
- This amplifier probe F can in turn contain a part F1 complementary to D3 and a nucleic acid-specific part F2.
- a splitting piece F ' is formed which, depending on the choice of sequence, has the same effect with splitting product D' or splitting product B '.
- An even higher degree of amplification is achieved when the slit piece is reinserted into an extension / disassembly cycle using additional template and amplification probes.
- the system according to the invention is therefore very flexible in the choice of the sequences of the nucleic acid-specific part in the probe nucleic acid or the amplifier probes.
- part D2 of the amplifier probe is complementary to C2 or a part thereof.
- hybrids of template nucleic acid C and the extension product of the cleavage product B 'and, if appropriate, the template nucleic acid C or E and the extension product of the cleavage product D' as well as any other hybrids can be separated, for example, by heat denaturation. In principle, however, non-thermal denaturing processes are also suitable.
- the extension of uncleaved amplifier probes e.g. B. on the extension products can be prevented analogously to probe nucleic acid B by blocking the 3 'end of the amplifier probe, for example by a dideoxynucleotide.
- the temperature of the method according to the invention is chosen so that the activities of the enzymes used are as optimal as possible and at the same time the hybridization conditions allow sufficient specificity.
- a temperature range between 30 ° and 60 °, particularly preferably 42 ° is expedient.
- thermostable RNAse higher temperatures are possible.
- the temperature also depends on the enzyme used for the extension. It is when using thermostable enzymes, for. B. Taq DNA polymerase a range between 50 ° and 80 ° is preferred and particularly preferably about 60-70 °.
- the template nucleic acid can also be a circular molecule.
- the use of an extension enzyme with strand displacement activity is preferred.
- the probe nucleic acid, the template nucleic acids and the amplifier nucleic acids can be produced by methods known in principle as soon as the sequence of their parts has been determined. For the preferred case that it is oligonucleotides with a length of less than 100 mononucleotide building blocks, the synthesis by common chemical methods (e.g. solid phase synthesis analogous to Merrifield) is preferred. This also enables the simple synthesis of mixed oligonucleotides (RNA / DNA chimera). For larger nucleic acids, recombinant production processes or chemical / enzymatic processes as described in EP-A-325970 are preferred. Part B1 is preferably 12-35, part B2 preferably 15-50, part C1 preferably 10-100 and part C2 15-50 nucleotides long. The same applies to the other template nucleic acids and the amplifier probes.
- FIG. 1 shows a basic embodiment of the method according to the invention, in which the part D2 of the amplifier probe is complementary to part C2 of the template nucleic acid, and thus the cleavage product D 'on the template nucleic acid C can be extended.
- FIG. 2 schematically shows an embodiment in which the cleavage product D 'is hybridized with a new template nucleic acid E.
- a probe nucleic acid F By using a probe nucleic acid F, a fragment F 'can be generated, which is either chosen so that it can hybridize with the template nucleic acid C (route 1), with the template nucleic acid E (route 2) or with another template nucleic acid G (route 3).
- Figures 3 and 4 show gels with the intermediates of the reaction.
- Figure 5 shows the amplification products in the gel.
- thermostable enzymes e.g. B. Thermus aquaticus polymerase and thermostable RNase H advantageous.
- the target nucleic acid A can be detected on the basis of one of the products or intermediate products formed in the amplification. It preferably takes place by detecting a label in at least one of the probe or template nucleic acids used or the cleavage or extension products formed. It is preferred that a template oligonucleotide is reacted with a cleavage product under suitable hybridization conditions together with a DNA polymerase and labeled deoxyribonucleoside triphosphates. The incorporation of labeled mononucleotides can be detected, for example, after the nucleic acids have been separated from unreacted labeled deoxyribonucleoside triphosphates. The use of two differently labeled nucleotides enables direct solid-phase detection.
- the template oligonucleotide will hybridize only with the chimeric RNA-DNA probe.
- the probe cannot be extended because the 3 'end does not hybridize to the template oligonucleotide.
- the method according to the invention provides a detection method in which both the extension product and the primers generated can be returned to the amplification cycle.
- an amplification cycle understood a reaction sequence in which a product of a reaction is in turn used to generate one or more of these products. In the drawings, such cycles are indicated by an arrow, which leads back to an earlier reaction stage. This results in an amplification with a theoretical amplification factor of more than 2 x if x is the number of cycles.
- the method according to the invention is superior to the methods based on PCR. In addition, it can be expanded to even higher amplification rates through a suitable design.
- the short nucleic acid sequences preferably represent the above-mentioned primers or cleavage products.
- the longer nucleic acid sequence is preferably a probe nucleic acid or amplifier nucleic acid.
- the extension area is therefore preferably the area D3.
- the breakdown of the longer nucleic acid preferably corresponds to the cleavage of the probe nucleic acid or the amplifier probe in part B1 or D1 or F1.
- the repetition of steps a) to c) preferably corresponds to the cycle management of the cleavage products.
- the amplifier probe D is split in part D1, a split product D 'being formed.
- this contains a sequence which enables hybridization to the template nucleic acid C and the extension analogous to the extension of B '. This would complete the amplification cycle.
- the amplifier probe D therefore contains, in addition to part D1, a nucleic acid-specific part which is homologous to B2 or a part thereof.
- nucleic acid-specific part B2 does not hybridize with the target nucleic acid if possible.
- the denaturation of the hybrid from the extension product of the cleavage product B 'with the template nucleic acid C preferably takes place between steps d) and e).
- the amplifier probe D is selected such that it contains, in addition to part D1, a nucleic acid-specific part D2 which, however, is not homologous to B2 or a part thereof.
- Part D2 is particularly preferably specific for part E2 of template nucleic acid E.
- the nucleic acid D preferably additionally contains a part D2 which is homologous to B2 or a part thereof.
- D2 is complementary to C2.
- part D1 is also complementary to B3.
- the sets according to the invention can also contain the reagents required for the detection, in particular degrading enzymes, e.g. B. RNAse H, a nucleic acid extension enzyme, e.g. B. contain a DNA polymerase or reverse transcriptase, mononucleotides and suitable for the enzyme reactions buffer.
- degrading enzymes e.g. B. RNAse H
- nucleic acid extension enzyme e.g. B. contain a DNA polymerase or reverse transcriptase, mononucleotides and suitable for the enzyme reactions buffer.
- RNA-DNA probe molecule B: 5'-GATCGGACTGGAAGTAATACGACTCACcgauacuaacauugagauucccg-3 ', SEQ.ID.NO. 1
- A 5'-ATCTCGGGAATCTCAATGTTAG.AT, SEG NO. 2
- buffer P2 10 mM Hepes, 1 mM MgCl2, pH 8.0
- the batches are mixed with 1 pmol each of the template oligonucleotide (C: 5'-CGACGCCGCGTCGCAGAAGATCGGTGAGTCGTATTACTTCCAGTCCGATC -3 ', SEQ.ID.NO. 3), heated to 100 ° C for 1 min and immediately cooled in ice. Then 3 ⁇ l 10 x Taq buffer (100 mM Tris pH 8.3, 500 mM KCl, 15 mM MgCl 2 , 1 mg / ml gelatin), dNTPs (final concentration of 1 mM each dATP, dCTP, dGTP, dTTP) are added to each batch.
- the template oligonucleotide C: 5'-CGACGCCGCGTCGCAGAAGATCGGTGAGTCGTATTACTTCCAGTCCGATC -3 ', SEQ.ID.NO. 3
- 3 ⁇ l 10 x Taq buffer 100 mM Tris pH 8.3, 500 mM KCl
- the oligonucleotides (including the accumulated, 5'-terminally radioactively labeled elongation products of the cleavage product D 'of the amplifier probe) are placed on a 12% sequence gel (Sambrook et al. (1989) Molecular Cloning, Cold Spring Harbor Laboratory Press, S. 6.36 ff.) And electrophoretically separated.
- the identification of the elongated primers D 'and thus the conclusion about the presence of the target sequence A is carried out by means of autoradiography.
- RNA-DNA probe molecule B: 5'-GATCGGACTGGAAGTAATACGACTCACcgauacuaacauugagauucccg-3 ', SEQ.ID NO. 1
- A 5'-ATCTCGGGAATCTCAATGTTAGNATOGQ.-3 2
- P2 10 mM Hepes, 1 mM MgCl2, pH 8.0
- the batches are each mixed with 10 pmol of the template oligonucleotide (C: 5'-CGACGCCGCGTCGCAGAAGATCGGTGAGTCGTATTACTTCCAGTCCGATC -3 ', SEQ.ID.NO. 3), heated to 100 ° C. for 1 min and immediately cooled in ice.
- C 5'-CGACGCCGCGTCGCAGAAGATCGGTGAGTCGTATTACTTCCAGTCCGATC -3 ', SEQ.ID.NO. 3
- the precipitated oligonucleotides (including the accumulated BIO-DIG-labeled elongation products) are each taken up in 210 ⁇ l buffer D (10 mM Hepes pH 8.3, 30 mM NaCl, 1 mM MnCl 2 ).
- the double-labeled synthesis products are immobilized in a streptavidin-coated microtiter plate (MTP) (streptavidin-coated microtiter plate from reverse transcriptase assay, non-radioactive, Boehringer Mannheim 1468 120) by placing two 100 ⁇ l portions of the oligonucleotide solutions in the wells of one with wash buffer (0.5% (v / v) Tween 20 in PBS ("phosphate buffered saline”)) MTP were pipetted. The MTP is incubated with shaking (Well-Warm1, Denley Instuments GmbH) for 1 h at 37 ° C.
- MTP streptavidin-coated microtiter plate
- the MTP with the immobilized nucleic acid molecules is washed five times with 200 ⁇ l wash buffer. Then 100 ⁇ l of the conjugate dilution (polyclonal ⁇ DIG ⁇ -S-Fab-PODpoly conjugate (Boehringer Mannheim GmbH), 200 mU / ml in conjugate buffer (100 mM Na phosphate pH 7.5, 0.9% (G / V) NaCl, 1% (w / v) Ralufon F4J or RSA fraction V; the conjugate buffer is treated with diethyl pyrocarbonate, sterile filtered (0.2 ⁇ m filter, Nalgene) and stored at 4 ° C)) and the MTP again under incubated under the same conditions.
- conjugate buffer 100 mM Na phosphate pH 7.5, 0.9% (G / V) NaCl, 1% (w / v) Ralufon F4J or RSA fraction V
- the conjugate buffer is treated with diethy
- RNA DNA probe molecule B 5'-GATCGGACTGGAAGTAATACGACTCACcgauacuaacauugagauucccmer-3', Oligo with 23 ribos at the 3 'end, SEQ.ID.NO.
- the samples were completely applied to 20% polyacrylamide gel (Biometra mini gel chamber).
- Running buffer 1 x TBE, running time approx. 1 hour at 250 V.
- FIG. 3 shows individual stages of the reaction (ethidium bromide staining)
- Figure 4 shows the reaction after blotting.
- Lane 1 25pmol B
- Lane 6 like 3 + 25pmol C ⁇ filling reaction
- Lane 2 20 pmol C.
- Lane 7 like 4 ⁇ filling reaction
- Lane 3 25pmol A + 25pmol B + RNase H
- Lane 8 like 5 + 25pmol C ⁇ filling reaction
- Lane 4 25pmol A + 25pmol B + RNase H
- Lane 9 25pmol C ⁇ filling reaction
- Lane 5 25pmol A + 25pmol B without nose H
- Lane 10 25pmol C + 25pmol A ⁇ replenishment reaction
- 2 ⁇ l of the eluted DNA was mixed with 20 pmol template C, 20 pmol amplifier probe (5'-GATCGGACTGGAAGTAATACGACTCAGcgccgcgucgcagaagauc-3 ', 46mer, DNA-RNA oligo with 19 ribos at the 3' end, SEQ.ID.NO. 4), 5 ⁇ l 10 x buffer (see above), each 100 ⁇ M dATP, dCTP and dGTP, 65 ⁇ M dTTP, 35 ⁇ M DIG-dUTP (each final concentration), 40 U RNasin, 5 U thermostable RNase H and 2.5 U Taq DNA polymerase made up to 50 ⁇ l in one batch. After a 3-minute denaturation step, the following cycle was repeated 10 times: 20 min. 42 ° C - 10 min. 60 ° C - 1 min. 95 ° C.
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DE4441626 | 1994-11-23 | ||
DE4441626A DE4441626A1 (de) | 1994-11-23 | 1994-11-23 | Verfahren zum besonders sensitiven Nachweis von Nukleinsäuren |
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EP0718408A2 true EP0718408A2 (fr) | 1996-06-26 |
EP0718408A3 EP0718408A3 (fr) | 1998-04-01 |
EP0718408B1 EP0718408B1 (fr) | 2003-08-13 |
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EP95118269A Expired - Lifetime EP0718408B1 (fr) | 1994-11-23 | 1995-11-21 | Méthode sensible pour détecter des acides nucléiques |
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US (1) | US5783392A (fr) |
EP (1) | EP0718408B1 (fr) |
JP (1) | JP2807202B2 (fr) |
AT (1) | ATE247174T1 (fr) |
CA (1) | CA2163587C (fr) |
DE (2) | DE4441626A1 (fr) |
ES (1) | ES2203628T3 (fr) |
FI (1) | FI114992B (fr) |
Cited By (3)
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EP0705905A2 (fr) * | 1994-07-16 | 1996-04-10 | Roche Diagnostics GmbH | Procédé pour la détection sensible d'acides nucléiques |
WO2020007904A1 (fr) | 2018-07-05 | 2020-01-09 | Bayer Aktiengesellschaft | Thiophènecarboxamides et analogues substitués utilisés en tant qu'agents antibactériens |
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US7076127B2 (en) * | 2003-01-14 | 2006-07-11 | Fuji Photo Film Co., Ltd. | Optical switch and safety apparatus using the same |
US7344834B2 (en) * | 2003-12-08 | 2008-03-18 | Cytyc Corporation | Method for DNA amplification using DNA blocking probes |
US20060115838A1 (en) * | 2004-10-19 | 2006-06-01 | Trevigen Inc. | Real-time detection of amplicons using nucleic acid repair enzymes |
KR100652903B1 (ko) * | 2005-12-21 | 2006-12-04 | 한국과학기술연구원 | 초흡수성 고분자를 함유한 제습제의 제조 방법 및 그 제조장치 |
NZ548731A (en) * | 2006-07-24 | 2008-12-24 | Zygem Corp Ltd | Isothermal detection methods and uses thereof |
WO2009133473A2 (fr) * | 2008-01-08 | 2009-11-05 | Zygem Corporation Limited | Méthodes de détection isothermes et utilisations de celles-ci |
MX2010010161A (es) * | 2008-03-15 | 2011-03-21 | Hologic Inc Star | Composiciones y metodos para el analisis de moleculas de acido nucleico durante reacciones de amplificacion. |
US20200263233A1 (en) * | 2017-06-23 | 2020-08-20 | Eiken Kagaku Kabushiki Kaisha | Method For Nucleic Acid Detection, Primer For Nucleic Acid Detection, And Kit For Nucleic Acid Detection |
KR20200054268A (ko) * | 2017-09-14 | 2020-05-19 | 알레레 샌디에고, 인크 | 이중-합텐 프로브를 이용한 재조합효소 폴리머라제 증폭의 검출 |
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WO1993019202A2 (fr) * | 1992-03-10 | 1993-09-30 | The United States Of America, As Represented By The Secretary, Department Of Health & Human Services | Reaction a matrice echangeable |
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US5011769A (en) * | 1985-12-05 | 1991-04-30 | Meiogenics U.S. Limited Partnership | Methods for detecting nucleic acid sequences |
IL86724A (en) * | 1987-06-19 | 1995-01-24 | Siska Diagnostics Inc | Methods and kits for amplification and testing of nucleic acid sequences |
US4994368A (en) * | 1987-07-23 | 1991-02-19 | Syntex (U.S.A.) Inc. | Amplification method for polynucleotide assays |
CA1340807C (fr) * | 1988-02-24 | 1999-11-02 | Lawrence T. Malek | Procede d'amplification d'une sequence d'acide nucleique |
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1994
- 1994-11-23 DE DE4441626A patent/DE4441626A1/de not_active Ceased
-
1995
- 1995-11-21 EP EP95118269A patent/EP0718408B1/fr not_active Expired - Lifetime
- 1995-11-21 AT AT95118269T patent/ATE247174T1/de active
- 1995-11-21 DE DE59510762T patent/DE59510762D1/de not_active Expired - Lifetime
- 1995-11-21 ES ES95118269T patent/ES2203628T3/es not_active Expired - Lifetime
- 1995-11-22 FI FI955619A patent/FI114992B/fi not_active IP Right Cessation
- 1995-11-22 US US08/561,632 patent/US5783392A/en not_active Expired - Lifetime
- 1995-11-22 JP JP7328052A patent/JP2807202B2/ja not_active Expired - Fee Related
- 1995-11-23 CA CA002163587A patent/CA2163587C/fr not_active Expired - Fee Related
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WO1993019202A2 (fr) * | 1992-03-10 | 1993-09-30 | The United States Of America, As Represented By The Secretary, Department Of Health & Human Services | Reaction a matrice echangeable |
DE4431269A1 (de) * | 1994-07-16 | 1996-01-18 | Boehringer Mannheim Gmbh | Verfahren zum sensitiven Nachweis von Nukleinsäuren |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0705905A2 (fr) * | 1994-07-16 | 1996-04-10 | Roche Diagnostics GmbH | Procédé pour la détection sensible d'acides nucléiques |
EP0705905A3 (fr) * | 1994-07-16 | 1999-03-03 | Roche Diagnostics GmbH | Procédé pour la détection sensible d'acides nucléiques |
WO2020007904A1 (fr) | 2018-07-05 | 2020-01-09 | Bayer Aktiengesellschaft | Thiophènecarboxamides et analogues substitués utilisés en tant qu'agents antibactériens |
WO2020079167A1 (fr) | 2018-10-18 | 2020-04-23 | Bayer Aktiengesellschaft | Hétéroarylaminoquinolines et analogues |
Also Published As
Publication number | Publication date |
---|---|
JPH08205894A (ja) | 1996-08-13 |
US5783392A (en) | 1998-07-21 |
FI114992B (fi) | 2005-02-15 |
EP0718408B1 (fr) | 2003-08-13 |
DE59510762D1 (de) | 2003-09-18 |
ATE247174T1 (de) | 2003-08-15 |
EP0718408A3 (fr) | 1998-04-01 |
CA2163587C (fr) | 2005-07-12 |
FI955619A (fi) | 1996-05-24 |
CA2163587A1 (fr) | 1996-05-24 |
DE4441626A1 (de) | 1996-05-30 |
ES2203628T3 (es) | 2004-04-16 |
FI955619A0 (fi) | 1995-11-22 |
JP2807202B2 (ja) | 1998-10-08 |
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